PROJECT SUMMARY/ABSTRACT Prostate-Specific Membrane Antigen (PSMA) is an 'ideal biomarker' for tumor targeting as it exhibits restricted expression on prostate tumor cells and the neovasculature of a number of other solid tumors. Cancer Targeted Technology has developed unique phosphoramidate-based PSMA inhibitor scaffolds that irreversibly bind to the enzyme target and selectively penetrate prostate tumor cells through the internalization of the PSMA enzyme-inhibitor complex, accumulating in endosomes and lysosomes. We recently demonstrated that the addition of an albumin-binding motif to our PSMA-targeted agents dramatically slows clearance in mice, resulting in tumor uptake of nearly 50% injected dose/g. Furthermore, CTT has licensed a novel pH-triggered linker system (Phos-Am) that is stable under physiological conditions, but can be tuned to rapidly release amine-containing drugs at endosomal/lysosomal pH. Together, these advances support the development of a PSMA-targeted drug-conjugate that can be trafficked to and selectively liberated within PSMA(+) tumor cells. The objective of this Phase-I application is to design and prove the effectiveness of a PSMA-targeted Small Molecule Drug Conjugate (SMDC): CTT1700. CTT1700 will incorporate these crucial components: phosphoramidate-based PSMA-targeting molecule, albumin binder, Phos-Am linker system, and Monomethyl Auristatin E (MMAE) as the conjugated drug payload. We expect that CTT1700 will exhibit the following key in vitro and in vivo performance characteristics: specificity for and rapid internalization by PSMA(+) prostate tumor cells, stability in plasma, rapid subcellular drug release, appropriate biodistribution and circulation half- life and potent anti-tumor activity. Our aims for this Phase-I application are as follows: Aim #1: Determine the stability and in vitro performance of CTT1700. We will prepare CTT1700 and assess its stability and drug release kinetics in buffer (pH = 4.5-7.4), as well as in mouse and human plasma. Concentration-dependent in vitro cytotoxicity and specificity will be assessed with PSMA(+) and PSMA(-) cells lines. Aim #2: Determine the in vivo efficacy of CTT1700. We will determine the biodistribution of CTT1700 and its released drug, MMAE, in blood and key organs. In vivo performance will be evaluated using a PSMA(+) tumor xenograft mouse model dosed once per week for 6 weeks and monitoring tumor volume for 10 weeks. This innovative work harnesses the ideal performance properties of a cell-penetrating irreversible small- molecule PSMA inhibitor that possesses the desirable pharmacokinetics of antibodies without the additional manufacturing costs associated with antibodies. Further, CTT1700 should have considerable significance for prostate cancer therapy by reducing chemotherapy-associated toxic side effects to non-target tissues. It is expected that CTT1700 will represent a breakthrough in targeted chemotherapy for prostate cancer and other tumors that are characterized by PSMA expression.